(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`International Bureau
`
`(43) International Publication Date
`
`
`(19) World Intellectual Property Organization
`fr?flhsk
`
` 25 August 2011 (25.08.2011) PCT W0 201 1/103300 A2
`
`
`(10) International Publication Number
`
`
`(51) International Patent Classification:
`C12N1/19 (2006.01)
`C12P 7/16 (2006.01)
`CIZN 15/52 (200601)
`G01N 33/68 (200601)
`nternatlona
`pp 1catlon um er:
`(21)I
`'
`1A 1'
`N
`b
`'
`PCT/US2011/025258
`
`[US/US]; 27 Piersons Ridge, Hockessin, Delaware
`W.
`19707 (US).
`I. du Pont de
`(74) Agent: LHULIER, Christine, M.; E.
`ecor 5
`enter,
`emours an
`ompany,
`ega
`atent
`R
`dC
`N
`dC
`L
`1P
`4417 Lancaster Pike, Wilmington, Delaware 19805 (US).
`
`(22) International Filing Date:
`
`17 February 2011 (17.02.2011)
`E 1. h
`“g 15
`English
`
`.
`25 FT L
`)
`1 mg anguage
`(
`(26) Publication Language:
`_
`_
`(30) Pmrlty Data:
`US
`17 February 2010 (17.02.2010)
`61/305,333
`(71) Applicant 0’or all designated States except US): BUTA-
`MAX(TM) ADVANCED BIOFUELS LLC [US/US];
`200 Powder Mill Road, Wilmington, Delaware 19803
`(US).
`Inventors; and
`Inventors/Applicants (for US only): FLINT, Dennis
`[US/US]; 31 Tenby Chase Drive, Newark, Delaware
`19711 (US). PAUL, Brian James [US/US]; 115 Fairfax
`Boulevard, Wilmington, Delaware 19803 (US). YE, Rick,
`
`(72)
`(75)
`
`(81) Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FL GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, N1,
`NO» NZ» OM» PE» PG» PH» PL» PT» RO» RS» RU» SC» SD»
`SE» SG» SK» SL» SM» ST» 5"» SY» TH» TJ» TM» TN» TR»
`TTaTzaUAaUGaUS»UzaVcaVNazAZMJW
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM» KE» LR» LS» MW» MZ» NA» SD» SL» SZ» TZ» UG»
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, 1T, LT, LU,
`Lv, MC, MK, MT, NL, NO, PL, PT, Ro, RS, SE, SI, SK,
`
`[Continued on nextpage]
`
`(54) Title: IMPROVING ACTIVITY OF FE-S CLUSTER REQUIRING PROTEINS
`
`Figure 1A
`
`FBAtR(Sa:l|)
`FBAterminaiar _
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`
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`_
`.
`FBAPramoter §
`'
`
`,
`
`2 micron
`
`g
`
`Figure 13
`
`pHR81 FBApiIvD(Sm)
`11i82 hp
`
`(57) Abstract: The present invention is related to a recombinant host cell,
`in particular a yeast cell, comprising a dihydroxy-acid dehydratase
`polypeptide. The invention is also related to a recombinant host cell having
`increased specific activity of the dihydroxy-acid dehydratase polypeptide
`as a result of increased expression of the polypeptide, modulation of the
`Fe-S cluster biosynthesis of the cell, or a combination thereof. The present
`invention also includes methods of using the host cells, as well as, methods
`for identifying polypeptides that
`increase the flux in an Fe-S cluster
`biosynthesis pathway in a host cell.
`
`pBR322 ori
`
`URA3
`
`ApR
`
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`R
`
`\
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`
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`
`,AaIH (In)
`
`. kg
`
`Delta
`
`8354M)
`’
`pZKDelta(s)- Leu2- FBAp- ilvD(Sm)‘ Afivmsm
`
`
`
`A551 (57b0
`Delta
`
`R
`M kgRR®
`FBAp
`Defective LEU2
`
`BUTAMAX 1003
`
`
`
`W02011/103300A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`

`

`WO 2011/103300 A2 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`Published:
`
`— without international search report and to be republished
`upon receipt ofthat report (Rule 48.2(g))
`
`— with sequence listing part ofdescription (Rule 52(0))
`
`

`

`WO 2011/103300
`
`PCT/US2011/025258
`
`IMPROVING ACTIVITY OF FE-S CLUSTER REQUIRING PROTEINS
`
`Cross-Reference to Related Applications
`
`[0001]
`
`This application claims the benefit of US. Provisional Appl. No. 61/305,333, filed
`
`February 17, 2010, which is incorporated by reference in its entirety.
`
`Sequence Listing Information
`
`[0002]
`
`The content of the electronically submitted sequence listing in ASCII text file
`
`CL4842sequencelisting.txt filed with the application is incorporated herein by reference
`
`in its entirety.
`
`BACKGROUND OF THE INVENTION
`
`Field of the Invention
`
`[0003]
`
`This invention relates generally to the fields of microbiology and biochemistry.
`
`Specifically, the present invention is related to a recombinant host cell, in particular a
`
`yeast cell, comprising a dihydroxy-acid dehydratase polypeptide. The invention is also
`
`related to a recombinant host cell having increased specific activity of the dihydroxy-acid
`
`dehydratase polypeptide as
`
`a result of increased expression of the polypeptide,
`
`modulation of the Fe-S cluster biosynthesis activity of the cell, or a combination thereof.
`
`The present invention also includes methods of using the host cells, as well as methods
`
`for identifying polypeptides that increase the flux in an Fe-S cluster biosynthesis pathway
`
`in a host cell.
`
`Background of the Invention
`
`[0004]
`
`Iron-sulfur (Fe-S) clusters serve as cofactors or prosthetic groups essential for the
`
`normal filnction of the class of proteins that contain them. In the class of Fe-S cluster
`
`containing proteins, the Fe-S clusters have been found to play several roles. When
`
`proteins of this class are first synthesized by the cell, they lack the Fe-S clusters required
`
`for their proper function and are referred to as apoproteins. Fe-S clusters are made in a
`
`series of reactions by proteins involved in Fe-S cluster biosynthesis and are transferred to
`
`the apo-proteins to form the filnctional Fe-S cluster containing holoproteins.
`
`

`

`W0 2011/103300
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`PCT/U82011/025258
`
`_ 2 _
`
`[0005]
`
`One such protein that requires Fe-S clusters for proper function is dihydroxy-acid
`
`dehydratase (DHAD)
`
`(EC. 4.2.1.9).
`
`DHAD catalyzes
`
`the conversion of 2,3-
`
`dihydroxyisovalerate to (x-ketoisovalerate, and of 2,3-dihydroxymethylvalerate to 0t-
`
`ketomethylvalerate. The DHAD enzyme is part of naturally occurring biosynthetic
`
`pathways producing the branched chain amino acids, (i.e., valine, isoleucine, leucine),
`
`and pantothenic
`
`acid
`
`(vitamin B5).
`
`DHAD catalyzed
`
`conversion of 2,3-
`
`dihydroxyisovalerate to (x-ketoisovalerate is also a common step in the multiple
`
`isobutanol biosynthetic pathways that are disclosed in US. Patent Appl. Pub. No. US
`
`20070092957 Al,
`
`incorporated by reference herein. Disclosed therein is, e.g.,
`
`the
`
`engineering of recombinant microorganisms for the production of isobutanol.
`
`[0006]
`
`High levels of DHAD activity are desired for increased production of products
`
`from biosynthetic pathways that include this enzyme activity, including, e.g., enhanced
`
`microbial production of branched chain amino acids, pantothenic acid, and isobutanol.
`
`Isobutanol, in particular, is useful as a filel additive, and its ready availability may reduce
`
`the demand for petrochemical fuels. However, since all known DHAD enzymes require a
`
`Fe-S cluster for their fianction, they must be expressed in a host having the genetic
`
`machinery to provide the Fe-S clusters required by these proteins. In yeast, mitochondria
`
`play an essential role in Fe-S cluster biosynthesis.
`
`If the DHAD is to be fianctionally
`
`expressed in yeast cytosol, a system to transport the requisite Fe-S precursor or signal
`
`from mitochondria and assemble the Fe-S cluster on the cytosolic apoprotein is required.
`
`Prior to the work of the present inventors, it was previously unknown whether yeast could
`
`provide Fe-S clusters for any DHAD located in the cytoplasm (since native yeast DHAD
`
`is located in the mitochondria) and more importantly when the DHAD is expressed at
`
`high levels in the cytoplasm
`
`[0007]
`
`Under certain conditions the rate of synthesis of Fe-S cluster requiring apo-
`
`proteins may exceed the cell's ability to synthesize and assemble Fe-S clusters for them.
`
`Cluster-less apo-proteins that accumulate under these conditions cannot carry out their
`
`normal function. Such conditions can include 1) the expression of a heterologous Fe-S
`
`cluster requiring protein especially in high amounts, 2) the expression of a native Fe-S
`
`cluster biosynthesis protein at higher levels than normal, or 3) a state where the host cell's
`
`ability to synthesize Fe-S clusters is debilitated.
`
`

`

`W0 2011/103300
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`PCT/U82011/025258
`
`_ 3 _
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0008]
`
`Disclosed herein is the surprising discovery that recombinant host cells expressing
`
`a high level of a heterologous Fe-S cluster requiring protein can supply the complement
`
`of Fe-S clusters for that protein if the level(s) of at least one Fe uptake, utilization, and/or
`
`Fe-S cluster biosynthesis protein are altered.
`
`[0009]
`
`Provided herein are recombinant host cells comprising at least one heterologous
`
`polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity
`
`wherein said at least one heterologous polynucleotide comprises a high copy number
`
`plasmid or a plasmid with a copy number that can be regulated. Also provided are
`
`recombinant host cells comprising at least one heterologous polynucleotide encoding a
`
`polypeptide having dihydroxy-acid dehydratase activity wherein said at
`
`least one
`
`heterologous polynucleotide is integrated at least once in the recombinant host cell DNA.
`
`Also provided are recombinant host cells comprising at
`
`least one heterologous
`
`polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity,
`
`wherein said host cell comprises at least one deletion, mutation, and/or substitution in an
`
`endogenous gene encoding a polypeptide affecting iron metabolism or Fe-S cluster
`
`biosynthesis. Also provided are recombinant host cells comprising at
`
`least one
`
`heterologous polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase
`
`activity and at least one heterologous polynucleotide encoding a polypeptide affecting
`
`iron metabolism or Fe-S cluster biosynthesis.
`
`[0010]
`
`In embodiments,
`
`said heterologous polynucleotide encoding a polypeptide
`
`affecting Fe-S cluster biosynthesis is selected from the group consisting of the genes in
`
`Tables 7, 8 and 9.
`
`In embodiments, said heterologous polynucleotide encoding a
`
`polypeptide affecting Fe-S cluster biosynthesis is selected from the group consisting of
`
`AFTl, AFT2, CCCl, FRA2, and GRX3, and combinations thereof.
`
`In embodiments,
`
`polypeptide is encoded by a polynucleotide that is constitutive mutant.
`
`In embodiments,
`
`said constitutive mutant is selected from the group consisting of AFTl L99A, AFTl
`
`LlOZA, AFTl C29lF, AFTl C293F, and combinations thereof.
`
`In embodiments said
`
`polypeptide affecting Fe-S cluster biosynthesis
`
`is encoded by a polynucleotide
`
`comprising a high copy number plasmid or a plasmid with a copy number that can be
`
`regulated.
`
`In embodiments, said polypeptide affecting Fe-S cluster biosynthesis is
`
`encoded by a polynucleotide integrated at least once in the recombinant host cell DNA.
`
`

`

`W0 2011/103300
`
`PCT/U82011/025258
`
`_ 4 _
`
`In embodiments, the at least one deletion, mutation, and/or substitution in an endogenous
`
`gene encoding a polypeptide affecting Fe-S cluster biosynthesis is selected from the
`
`group consisting of CCCl, FRA2, and GRX3, and combinations
`
`thereof.
`
`In
`
`embodiments,
`
`the at
`
`least one heterologous polynucleotide encoding a polypeptide
`
`affecting Fe-S cluster biosynthesis is selected from the group consisting of AFTl, AFT2,
`
`their mutants, and combinations thereof
`
`[0011]
`
`In embodiments, said at
`
`least one heterologous polynucleotide encoding a
`
`polypeptide having dihydroxy-acid dehydratase activity is expressed in multiple copies.
`
`In embodiments, said at least one heterologous polynucleotide comprises a high copy
`
`number plasmid or a plasmid with a copy number that can be regulated. In embodiments,
`
`said at least one heterologous polynucleotide is integrated at least once in the recombinant
`
`host cell DNA.
`
`In embodiments, said Fe-S cluster biosynthesis is increased compared to
`
`a recombinant host cell having endogenous Fe-S cluster biosynthesis.
`
`[0012]
`
`In embodiments, said host cell is a yeast host cell.
`
`In embodiments, said yeast
`
`host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces,
`
`Hansenula, Candida, Kluyvemmyces, Yarrowz'a, Issatchenkz'a and Pichia.
`
`[0013]
`
`In
`
`embodiments,
`
`said
`
`heterologous
`
`polypeptide
`
`having
`
`dihydroxy-acid
`
`dehydratase activity is expressed in the cytosol of the host cell.
`
`In embodiments, said
`
`heterologous polypeptide having dihydroxy-acid dehydratase activity has an amino acid
`
`sequence that matches the Profile HMM of Table 12 with an E value of < 10'5 wherein
`
`the polypeptide fiarther comprises all
`
`three conserved cysteines, corresponding to
`
`positions 56, 129, and 201 in the amino acids sequences of the Streptococcus mutans
`
`DHAD enzyme corresponding to SEQ ID NO:168.
`
`In embodiments, said heterologous
`
`polypeptide having dihydroxy-acid dehydratase activity has an amino acid sequence with
`
`at least about 90% identity to SEQ ID NO: 168 or SEQ ID NO: 232.
`
`In embodiments
`
`said polypeptide having dihydroxy-acid dehydratase activity has a specific activity
`
`selected from the group consisting of:
`
`greater than about 5-fold with respect to the
`
`control host cell comprising at
`
`least one heterologous polynucleotide encoding a
`
`polypeptide having dihydroxy-acid dehydratase activity, greater than about 8-fold with
`
`respect to the control host cell comprising at
`
`least one heterologous polynucleotide
`
`encoding a polypeptide having dihydroxy-acid dehydratase activity, or greater than about
`
`10-fold with respect
`
`to the control host cell comprising at
`
`least one heterologous
`
`

`

`WO 2011/103300
`
`PCT/US2011/025258
`
`_ 5 _
`
`polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity.
`
`In
`
`embodiments said polypeptide having dihydroxy-acid dehydratase activity has a specific
`
`activity selected from the group consisting of: greater than about 3-fold with respect to a
`
`control host cell comprising at
`
`least one heterologous polynucleotide encoding a
`
`polypeptide having dihydroxy-acid dehydratase activity and greater than about 6-fold
`
`with respect to the control host cell comprising at least one heterologous polynucleotide
`
`encoding a polypeptide having dihydroxy-acid dehydratase activity.
`
`In embodiments,
`
`said polypeptide having dihydroxy-acid dehydratase activity has a specific activity
`
`selected from the group consisting of: greater than about 0.25 U/mg; greater than about
`
`0.3 U/mg; greater than about 0.5 U/mg; greater than about 1.0 U/mg; greater than about
`
`1.5 U/mg; greater than about 2.0 U/mg; greater than about 3.0 U/mg; greater than about
`
`4.0 U/mg; greater than about 5.0 U/mg; greater than about 6.0 U/mg; greater than about
`
`7.0 U/mg; greater than about 8.0 U/mg; greater than about 9.0 U/mg; greater than about
`
`10.0 U/mg; greater than about 20.0 U/mg; and greater than about 50.0 U/mg.
`
`[0014]
`
`In embodiments
`
`said recombinant host cell produces
`
`isobutanol, and in
`
`embodiments, said recombinant host cell comprises an isobutanol biosynthetic pathway.
`
`[0015]
`
`Also provided herein are methods of making a product comprising: providing a
`
`recombinant host cell; and contacting the recombinant host cell of with a fermentable
`
`carbon substrate in a fermentation medium under conditions wherein said product is
`
`produced;, wherein the product is selected from the group consisting of branched chain
`
`amino acids, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol, and
`
`combinations thereof.
`
`In embodiments,
`
`the methods further comprise optionally
`
`recovering said product.
`
`In embodiments, the methods further comprise recovering said
`
`product.
`
`[0016]
`
`Also provided are methods of making isobutanol comprising: providing a
`
`recombinant host cell; contacting the recombinant host cell with a fermentable carbon
`
`substrate in a fermentation medium under conditions wherein isobutanol is produced.
`
`In
`
`embodiments, the methods further comprise optionally recovering said isobutanol.
`
`In
`
`embodiments, the methods further comprise recovering said isobutanol.
`
`[0017]
`
`Also provided are methods for the conversion of 2,3-dihydroxyisovalerate to 0t-
`
`ketoisovalerate comprising: providing a recombinant host cell; growing the recombinant
`
`

`

`W0 2011/103300
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`PCT/US2011/025258
`
`_ 6 _
`
`host cell of under conditions where the 2,3-dihydroxyisovalerate is converted to 0L-
`
`ketoisovalerate.
`
`In embodiments,
`
`the conversion of 2,3-dihydroxyisovalerate to 0t-
`
`ketoisovalerate compared to a control host cell comprising at least one heterologous
`
`polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity is
`
`increased in an amount selected from the group consisting of: (a) at least about 5%; (b) at
`
`least about 10%; (c) at least about 15%; (d) at least about 20%; (e) at least about 25%; (i)
`
`at least about 30%; (g) at least about 35%; (h) at least about 40%; (i) at least about 45%;
`
`(j) at least about 50%; (k) at least about 60%; (l) at least about 70%; (m) at least about
`
`80%; (n) at least about 90%; and (0) at least about 95%.
`
`[0018]
`
`Also provided are methods for increasing the specific activity of a heterologous
`
`polypeptide having dihydroxy-acid dehydratase activity in a recombinant host cell
`
`comprising: providing a recombinant host cell; and growing the recombinant host cell of
`
`under
`
`conditions whereby the heterologous polypeptide having dihydroxy-acid
`
`dehydratase activity is expressed in fianctional form having a specific activity greater than
`
`the same host cell lacking said heterologous polypeptide.
`
`[0019]
`
`Also provided are methods for increasing the flux in an Fe-S cluster biosynthesis
`
`pathway in a host cell comprising: providing a recombinant host cell; and growing the
`
`recombinant host cell under conditions whereby the flux in the Fe-S cluster biosynthesis
`
`pathway in the host cell is increased.
`
`[0020]
`
`Also provide are methods of increasing the activity of an Fe-S cluster requiring
`
`protein in a recombinant host cell comprising: providing a recombinant host cell
`
`comprising an Fe-S cluster requiring protein; changing the expression or activity of a
`
`polypeptide affecting Fe-S cluster biosynthesis in said host cell; and growing the
`
`recombinant host cell under conditions whereby the activity of the Fe-S cluster requiring
`
`protein is increased. In embodiments, said increase in activity is an amount selected from
`
`the group consisting of: greater than about 10%; greater than about 20%; greater than
`
`about 30%; greater than about 40%; greater than about 50%; greater than about 60%;
`
`greater than about 70%; greater than about 80%; greater than about 90%; and greater than
`
`about 95%, 98%, or 99%.
`
`In embodiments, the increase in activity is in an amount
`
`selected from the group consisting of: greater than about 5-fold; greater than about 8-fold;
`
`greater than about 10-fold.
`
`In embodiments, the increase in activity is in an amount
`
`

`

`WO 2011/103300
`
`PCT/US2011/025258
`
`_ 7 _
`
`selected from the group consisting of: greater than about 3-fold and greater than about 6-
`
`fold.
`
`[0021]
`
`A method for identifying polypeptides that increase the flux in an Fe-S cluster
`
`biosynthesis pathway in a host cell comprising: changing the expression or activity of a
`
`polypeptide affecting Fe-S cluster biosynthesis; measuring the activity of a heterologous
`
`Fe-S cluster requiring protein; and comparing the activity of the heterologous Fe-S cluster
`
`requiring protein measured in the presence of the change in expression or activity of a
`
`polypeptide to the activity of the heterologous Fe-S cluster requiring protein measured in
`
`the absence of the change in expression or activity of a polypeptide, wherein an increase
`
`in the activity of the heterologous Fe-S cluster requiring protein indicates an increase in
`
`the flux in said Fe-S cluster biosynthesis pathway.
`
`[0022]
`
`Provided herein are methods for identifying polypeptides that increase the flux in
`
`an Fe-S cluster biosynthesis pathway in a host cell comprising: changing the expression
`
`or activity of a polypeptide affecting Fe-S cluster biosynthesis; measuring the activity of a
`
`polypeptide having dihydroxy-acid dehydratase activity; and comparing the activity of the
`
`polypeptide having dihydroxy-acid dehydratase activity measured in the presence of the
`
`change to the activity of the polypeptide having dihydroxy-acid dehydratase activity
`
`measured in the absence of change, wherein an increase in the activity of the polypeptide
`
`having dihydroxy-acid dehydratase activity indicates an increase in the flux in said Fe-S
`
`cluster biosynthesis pathway.
`
`[0023]
`
`In embodiments, said changing the expression or activity of a polypeptide
`
`affecting Fe-S cluster biosynthesis comprises deleting, mutating, substituting, expressing,
`
`up-regulating, down-regulating, altering the cellular location, altering the state of the
`
`protein, and/or adding a cofactor.
`
`In embodiments, the Fe-S cluster requiring protein has
`
`dihydroxy-acid dehydratase activity and wherein said Fe-S cluster requiring protein
`
`having dihydroxy-acid dehydratase activity has an amino acid sequence that matches the
`
`Profile HMM of Table 12 with an E value of < 10'5 wherein the polypeptide further
`
`comprises all three conserved cysteines, corresponding to positions 56, 129, and 201 in
`
`the amino acids sequences of the Streptococcus mutans DHAD enzyme corresponding to
`
`SEQ ID NO:l68.
`
`In embodiments, the polypeptide affecting Fe-S cluster biosynthesis is
`
`selected from the group consisting of the genes in Tables 7, 8 and 9.
`
`

`

`W0 2011/103300
`
`PCT/U82011/025258
`
`_ 8 _
`
`[0024]
`
`Also provided are recombinant host cells comprising at least one polynucleotide
`
`encoding a polypeptide identified by the methods provided herein.
`
`In embodiments, said
`
`host cell
`
`further comprises at
`
`least one heterologous polynucleotide encoding a
`
`polypeptide having dihydroxy-acid dehydratase
`
`activity.
`
`In embodiments,
`
`said
`
`heterologous polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase
`
`activity is
`
`expressed in multiple
`
`copies.
`
`In embodiments,
`
`said heterologous
`
`polynucleotide comprises a high copy number plasmid or a plasmid with a copy number
`
`that can be regulated.
`
`In embodiments, said heterologous polynucleotide is integrated at
`
`least once in the recombinant host cell DNA.
`
`[0025]
`
`In embodiments, said host cell is a yeast host cell.
`
`In embodiments, said yeast
`
`host cell is selected from the group consisting of Saccharomyces, Schizosaccharomyces,
`
`Hansenula, Candida, Kluyveromyces, Yarrowz'a,
`
`Issatchenkz'a,
`
`and Pichia.
`
`In
`
`embodiments, said heterologous polypeptide having dihydroxy-acid dehydratase activity
`
`is expressed in the cytosol of the host cell.
`
`In embodiments, said heterologous
`
`polypeptide having dihydroxy-acid dehydratase activity has an amino acid sequence that
`
`matches the Profile HMM of Table 12 with an E value of < 10'5 wherein the polypeptide
`
`further comprises all three conserved cysteines, corresponding to positions 56, 129, and
`
`201 in the amino acids sequences of the Streptococcus mutans DHAD enzyme
`
`corresponding to SEQ ID NO: 168.
`
`In embodiments, said recombinant host cell produces
`
`a product selected from the group consisting of branched chain amino acids, pantothenic
`
`acid, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol, and combinations thereof.
`
`In
`
`embodiments,
`
`recombinant host cell produces isobutanol.
`
`In embodiments,
`
`said
`
`recombinant host cell comprises an isobutanol biosynthetic pathway.
`
`In embodiments
`
`said isobutanol biosynthetic pathway comprises at least one polypeptide encoded by a
`
`polynucleotide heterologous
`
`to the host cell.
`
`In embodiments,
`
`said isobutanol
`
`biosynthetic pathway comprises at least two polypeptides encoded by polynucleotides
`
`heterologous to the host cell.
`
`[0026]
`
`In embodiments, monomers of the polypeptides of the invention having
`
`dihydroxy-acid dehydratase activity have an Fe-S cluster loading selected from the group
`
`consisting of: (a) at least about 10%; (b) at least about 15%; (c) at least about 20%; (d) at
`
`least about 25%; (e) at least about 30%; (f) at least about 35%; (g) at least about 40%; (h)
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`at least about 45%; (i) at least about 50%; (j) at least about 60%; (k) at least about 70%;
`
`(l) at least about 80%; (m) at least about 90%; and (n) at least about 95%.
`
`BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
`
`[0027]
`
`Figure 1A depicts a vector map of a vector for overexpression of the IZVD gene
`
`from S. mutans.
`
`[0028]
`
`Figure 1B depicts a vector map of an integration vector for overexpression of the
`
`IZVD gene from S. mutans in the chromosome.
`
`[0029]
`
`Figure 2 depicts a vector map of a centromere vector used to clone AFTI or AFTI
`
`mutants and useful for other genes of interest.
`
`[0030]
`
`[0031]
`
`[0032]
`
`[0033]
`
`[0034]
`
`[0035]
`
`[0036]
`
`[0037]
`
`[0038]
`
`[0039]
`
`Figure 3 depicts a UV-Vis absorbance spectrum of purified S. mutans DHAD.
`
`Figure 4 depicts an EPR spectrum of purified S. mutans DHAD.
`
`Figure 5 depicts a biosynthetic pathway for biosynthesis of isobutanol.
`
`Figure 6A depicts a schematic of Azotobacter vinelandz'z' m'f genes.
`
`Figure 6B depicts a schematic of additional Azotobacter vinelandz'z' m'fgenes.
`
`Figure 6C depicts a schematic of the equation in which NFU acts as a persulfide
`
`reductase.
`
`Figure 7 depicts a schematic of Helicobacter pylori m'f genes.
`
`Figure 8 depicts a schematic of E. coli isc genes.
`
`Figure 9 depicts a schematic of E. coli sufgenes.
`
`Figure 10 depicts a schematic of the cytosolic [2Fe-ZS] biosynthesis and assembly
`
`system.
`
`[0040]
`
`Figure ll depicts a vector map of a vector for overexpression of the IZVD gene
`
`from L. lactis.
`
`[0041]
`
`Table 12 is a table of the Profile HMM for dihydroxy-acid dehydratases based on
`
`enzymes with assayed function prepared as described in US. Patent Appl. No.
`
`12/569,636, filed Sept. 29, 2009. Table 12 is submitted herewith electronically and is
`
`incorporated herein by reference.
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`DETAILED DESCRIPTION OF THE INVENTION
`
`[0042]
`
`Described herein is a method to increase the fraction of the Fe-S cluster requiring
`
`proteins that are loaded with Fe-S clusters. Also described are recombinant host cells that
`
`express functional Fe-S cluster requiring proteins, such as DHAD enzymes, and at least
`
`one heterologous Fe uptake, utilization, or Fe-S cluster biosynthesis protein, recombinant
`
`host cells that express functional DHAD enzymes and comprise at least one deletion,
`
`mutation, and/or substitution in a native protein involved in Fe utilization or Fe-S cluster
`
`biosynthesis, or recombinant host cells comprising combinations thereof. In addition, the
`
`present invention describes a method to identify polypeptides that increase the flux in an
`
`Fe-S cluster biosynthesis pathway in a host cell. Also described is a method to identify
`
`polypeptides that alter the activity of an Fe-S cluster requiring protein.
`
`Definitions
`
`Unless defined otherwise, all technical and scientific terms used herein have the
`
`same meaning as commonly understood by one of ordinary skill in the art to which this
`
`invention belongs.
`
`In case of conflict, the present application including the definitions
`
`will control. Also, unless otherwise required by context, singular terms shall include
`
`pluralities and plural terms shall include the singular. All publications, patents and other
`
`references mentioned herein are incorporated by reference in their entireties for all
`
`purposes.
`
`[0043]
`
`[0044]
`
`[0045]
`
`In order to further define this invention, the following terms and definitions are
`
`herein provided.
`
`[0046]
`
`As used herein,
`
`the terms "comprises," "comprising," "includes," "including,"
`
`"has," "having,
`
`contains" or "containing," or any other variation thereof, will be
`
`understood to imply the inclusion of a stated integer or group of integers but not the
`
`exclusion of any other integer or group of integers. For example, a composition, a
`
`mixture, a process, a method, an article, or an apparatus that comprises a list of elements
`
`is not necessarily limited to only those elements but may include other elements not
`
`expressly listed or inherent to such composition, mixture, process, method, article, or
`
`apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or
`
`and not to an exclusive or. For example, a condition A or B is satisfied by any one of the
`
`following: A is true (or present) and B is false (or not present), A is false (or not present)
`
`and B is true (or present), and both A and B are true (or present).
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`[0047]
`
`As used herein,
`
`the term "consists of," or variations such as "consist of' or
`
`"consisting of," as used throughout the specification and claims, indicate the inclusion of
`
`any recited integer or group of integers, but that no additional integer or group of integers
`
`may be added to the specified method, structure, or composition.
`
`[0048]
`
`As used herein, the term "consists essentially of," or variations such as “consist
`
`essentially of” or "consisting essentially of," as used throughout the specification and
`
`claims, indicate the inclusion of any recited integer or group of integers, and the optional
`
`inclusion of any recited integer or group of integers that do not materially change the
`
`basic or novel properties of the specified method, structure or composition. See M.P.E.P.
`
`§ 21 l 1.03.
`
`[0049]
`
`Also, the indefinite articles "a" and "an" preceding an element or component of
`
`the invention are intended to be nonrestrictive regarding the number of instances, z'.e.,
`
`occurrences of the element or component. Therefore "a" or "an" should be read to include
`
`one or at least one, and the singular word form of the element or component also includes
`
`the plural unless the number is obviously meant to be singular.
`
`[0050]
`
`The term "invention" or "present invention" as used herein is a non-limiting term
`
`and is not intended to refer to any single embodiment of the particular invention but
`
`encompasses all possible embodiments as described in the application.
`
`[0051]
`
`As used herein,
`
`the term "abou " modifying the quantity of an ingredient or
`
`reactant of the invention employed refers to variation in the numerical quantity that can
`
`occur, for example, through typical measuring and liquid handling procedures used for
`
`making concentrates or solutions in the real world; through inadvertent error in these
`
`procedures; through differences in the manufacture, source, or purity of the ingredients
`
`employed to make the compositions or to carry out the methods; and the like. The term
`
`"about" also encompasses amounts that differ due to different equilibrium conditions for a
`
`composition resulting from a particular initial mixture. Whether or not modified by the
`
`term "about", the claims include equivalents to the quantities.
`
`In one embodiment, the
`
`term "about" means Within 10% of the reported numerical value, preferably Within 5% of
`
`the reported numerical value.
`
`[0052]
`
`The term "isobutanol biosynthetic pathway" refers to an enzyme pathway to
`
`produce isobutanol from pyruvate.
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`[0053]
`
`The term "a facultative anaerobe" refers to a microorganism that can grow in both
`
`aerobic and anaerobic environments.
`
`[0054]
`
`The term "carbon substrate" or "fermentable carbon substrate" refers to a carbon
`
`source capable of being metabolized by host organisms of the present invention and
`
`particularly carbon sources selected from the group consisting of monosaccharides,
`
`oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.
`
`[0055]
`
`The term "Fe-S cluster biosynthesis” refers to biosynthesis of Fe-S clusters,
`
`including, e.g.,
`
`the assembly and loading of Fe-S clusters. The term "Fe-S cluster
`
`biosynthesis genes", "Fe-S cluster biosynthesis proteins" or "Fe-S cluster biosynthesis
`
`pathway" refers to those polynucleotides/genes and the encoded polypeptides that are
`
`involved in the biosynthesis of Fe-S clusters, including, e. g., the assembly and loading of
`
`Fe-S clusters.
`
`[0056]
`
`The term “Fe uptake and utilization” refers to processes which can effect Fe-S
`
`cluster biosynthesis such as Fe sensing, uptake, utilization, and homeostasis. “Fe uptake
`
`and utilization genes” refers to those polynucleotides/genes and the encoded polypeptides
`
`that are involved in Fe uptake, utilization, and homeostasis.
`
`Some of these
`
`polynucleotides/genes are contained in the "Fe Regulon" that has bee